Diarrheal diseases are an important cause of death in infants and young children in developing countries. Diarrheal diseases are also an important cause of serious illness in the elderly or immune compromised adults, as well as in travelers to countries where sanitation is inadequate and where these diseases are endemic.
Enterotoxigenic Escherichia coli strains (ETEC) are a major cause of diarrheal disease. Accordingly, there is a need for a vaccine for preventing the diseases associated with these bacteria. Ideally, a vaccine generates a protective immune response in which it interferes with a very early step in the infectious process, for example, by preventing the attachment of the pathogenic bacterial cells to host tissue. In the case of ETEC, this attachment step is believed to be mediated by pili, which are long, proteinaceous structures extending out from the surface of the bacterial cells. The CS2 pili are believed to mediate attachment to and/or promote colonization of the human upper intestine.
Different human ETEC strains produce a number of serologically different types of pili. The pili differ in antigenic specificity and subunit (pilin) molecular weight. Among human ETEC strains there are CFA/I (colonization factor antigen 1), CS1 (coli surface antigen 1) and CS2 (coli surface antigen 2) Evans and Evans (1978) Infect. Immun. 21:638-647; Smyth (1982) J. Gen. Microbiol. 128:2081-2096!. The latter two were initially grouped together as CFA/II, a term that also includes the fibrillar CS3 surface structures Levine et al. (1984) Infect. Immun. 44:409-420!. These three pili types seem to form a family based on several kinds of data. Although present in different ETEC strains, they are all positively regulated by rns or a closely related gene Caron et al. (1989) Proc. Natl. Acad. Sci. USA 86:963-967; Caron et al. (1990) Infect. Immun. 58:3442-3444; Savelkoul et al. (1990) Microb. Pathogen. 8:91-99!. Second, the N-terminal amino acid sequences of the major pilin proteins are almost identical for these three pili types and for CS4 and PCF1066 Wolf et al. (1989) Infect. Immun. 57:164-173; Sommerfelt et al. (1992) Infect. Immun. 69:3799-3806!. Third, for CFA/I and CS1, the DNA sequences of the four genes required for pili expression show significant homology Hamers et al. (1989) Microbial Pathogenesis 6:297-309; Perez-Casal et al. (1990) Infect. Immun. 58:3594-3600; Froehlich et al. (1994) Molec. Microbiol. 12:387-401; Jordi et al. (1991) FEMS Microbiol. Lett. 80:265-270; Jordi et al. (1992) DNA Sequence 2:257-263!. Additionally, both CS1 and CFA/I are determined by plasmid-borne genes.
Only the genes encoding CS1 have been analyzed for function. The morphogenesis of CS1 pili appears to differ from that of other pili in Gram negative bacteria, for example, the pap pili of the E. coli strains associated with pyelonephritis or the common type 1 pili. Only four genes are needed to express CS1 pili in E. coli K12 Froehlich et al. (1994) supra!. The major pilin subunit, encoded by cooA, lacks the features of disulfide bonds and penultimate tyrosine residues common to many other pilins. Additionally, the CS1 and CFA/I proteins show no homology with any other known or predicted proteins in the databank Scott et al. (1992) Molec. Microbiol. 6:293-300; Froehlich et al. (1994) Molec. Microbiol. 12:387-401!. Thus, no typical chaperonins can be identified on the basis of sequence comparisons. Therefore, the pili on human ETEC strains are believed to belong to a unique family of pilins.
Mutations in any one of cooA, cooB, cooC and cooD result in an absence of pili. Only mutation in cooA prevents pilin synthesis (Scott et al. (1992) supra; Froehlich et al. (1994) supra!. There is no candidate for a pilus-associated adhesion other than the pilin protein itself.
There is a long felt need in the art for vaccines useful in protecting against diarrheal disease resulting from infection with ETEC, including CS2 ETEC. Because of the serological diversity in pili of ETEC, an ETEC vaccine advantageously includes antigenic determinants (epitopes) from more than one pilus type to be effective against more than one type of ETEC infection, or a single vaccine can include one or more conserved antigenic determinants.